Vibrio cholerae vaccine candidates engineered by deleting the ctx gene still show various degrees of residual reactogenicity, particularly diarrhea. Studies that we have undertaken have led to the discovery of a new enterotoxic determinant potentially involved in the pathogenesis of cholera, that acts by altering the structure of the intestinal zonula occludens (ZO). Because of the striking effect on the ZOI, we have designated the vibrio factor responsible for this effect as ZOT (for Zonula Occludens Toxin). This toxin may represent the archetype of a new family of enterotoxins that are responsible for a completely new diarrheagenic mechanism in infectious diarrhea. The long-term objectives of the proposed project are to further characterize the mechanism of action of this moiety and, by using ZOT as a ZO modulator, to gain insight into the pathophysiology of tight junction regulation, particularly at the cellular and molecular levels. Specifically we propose: AIM 1. To identify the receptor for ZOT. Preliminary data suggest that ZOT modifies intestinal tight junctions via an intracellular signal pathway, rather than acting directly on the paracellular junctional complex. We now intend to identify the enterocyte receptor specific for ZOT and to study its distribution throughout the intestine by using Maltose Binding Protein (MBP)-ZOT fusion protein. AIM 2. To determine whether ZOT regulates tight junction integrity through protein kinases C-mediated phosphorylation of relevant target protein within the tj complex and/or actin cytoskeleton. Our preliminary data suggest that the ZOT effect on actin reorganization and tissue permeability is mediated by PKC. We now intend to identify the tight junctional and/or actin cytoskeletal elements that are phosphorylated by ZOT-activated PKC. AIM 3. To determine whether ZOT regulates tissue permeability through actin reorganization and/or tight junction disassembly. In this studies we will determine the temporal relationship between actin reorganization, PKC activation, target protein phosphorylation, and tight junction permeability. AIM 4. To establish the mechanism of diarrheogenicity of ZOT. We have proven that ZOT increases intestinal tissue conductance by reversibly opening intercellular tight junctions. We now intend to study the effect of ZOT on the regulation of water and electrolyte transport via the paracellular pathway, both in vivo and in vitro.